The objectives of this proposal are to explore and further characterize the Arc two-component signal-transduction system of Escherichia coli, which modulates gene expression in response to shifts between respiratory and fermentative energy metabolism. This two-component system comprises the ArcB protein, a complex membrane sensor kinase having three cytosolic domains, and the ArcA protein, the response regulator controlling over 20 operons. Experiments are planned to elucidate several aspects of this system.(l) Different strategies will be tried to identify the redox signal and to define its ArcB reception site which can trigger the phospho-relay reaction that culminates in the conversion of ArcA to its phosphorylated and functional state. (2) The His -> Asp -> His -> Asp phospho-relay for signal amplification and the process for signal decay will be further characterized. (3) The mode of action of two effectors, modifying ArcB activity, and the regulation of their intracellular levels will be studied. The ArcB work will include comparisons with the simpler counterpart protein of the pathogen Haemophilus influenzae which has merely a transmitter domain. (4) Regulation of the ArcB-independent phosphorylation of ArcA by acetyl phosphate and the scope of ArcA-P action will be explored. Accomplishment of the goals should not only clarify the mechanisms of Arc signal-transduction and its scope of control, but could also broaden our understanding of the integration of genetic circuits for bacterial adaptation to environmental changes, including those associated with host invasion during pathogenesis. Gaining more precise information on two-component systems, abundant in bacteria but not found in mammals, could also facilitate rational screening for novel drugs to treat infections.